Cell biology and biomedical and pharmacological/biotechnological research and production that are dependent thereon are increasing in importance. This is embedded in research into gene and protein characterization, influenced by the Human Genome Project. This now also includes the creation and analysis of siRNA libraries (cf. for example Pepperkok et al., Nat Rev Mol Cell Biol 2006; 7: 690-696, High-Throughput Fluorescence Microscopy for Systems Biology) connected with methods for high throughput with automated imaging, optical methods and dispensing.
Several, already existing modules can be defined for this automated image-based high throughput for cell evaluation. To date, a core element of high throughput is automated micrography. This also includes image analysis with the corresponding data management (cf. for example the product Opera from the company Evotec Technologies). Another module can comprise various aspects of fluidic manipulation. This means for example the addition/dispensing of further agents, changing of media etc. For working with cells, the manipulation of cell culture vessels, so-called multi-plates such as 96-well and 384-well plates is essential. This means automated plate changing. In order to keep the cells as viable as possible even for longer-lasting analysis of quite large quantities of plates, various designs of a climate-control module are also offered by commercial suppliers. Meanwhile, some suppliers have developed automated systems, in particular for cultivating adherent animal cells and evaluating them by microscopy. The highly complex systems will not be explained in detail here, as they are commercially available (see for example Terstegge et al., Nature Methods 1, 271-272, 2004, Hamilton's new cellhost system for full automation of embryonic stem cell cultures). As a result of analysis, optical (mainly image-based) properties of the biological sample are used in particular for assessment, and features such as pH changes and ion-selective change of the medium only to a small extent.
A technique originally designed for the analysis of individual cells is the classical patch-clamp technique. For the screening of active substances, later on automatic systems were developed, which automate measurement and rinsing procedures and in addition are operated in parallel in a modified planar structure (cf. for example the description of the MDC PatchXpress 7000A, 16-Channel Automated Patch Clamp System, http://www.chantest.com/fastpatch.html). Manual patching is the gold standard—with sensible protocols there are hardly any false-negatives and false-positives. With planar patching, the sensitivity is reduced and the deviation can be at a factor of three, and even more with increased automation, and additionally false-negative results in particular may be increased. Therefore, in the search for active substances, various patch techniques—varying in price, throughput, and accuracy of the information obtained—are employed in the preliminary, main, and final phases of measurement campaigns.
Automated systems and automated imaging techniques for investigating cell-biology processes are known, and are used in the pharmacological, medical and cell-biology research and analysis of active substances including substance screening. Typically, fluorescence-optical properties are evaluated, such as change in intensity of fluorescence, distribution of fluorescence or a shift of fluorescence, which can for example result from energy transport (FRET: fluorescence resonance energy transfer). Measured values are recorded both by confocal and non-confocal techniques. Camera-based image recording is very quick. Depending on fluorescence labelling, camera sensitivity, the objective lens, the resolution and the excitation intensity, typically about 2 to about 100 cells with a cell size of about 10×10 μm2 and a cell height of 5 μm are recorded simultaneously at an exposure of for example 40 ms. This speed generally also allows recording of images in different z-heights (z-stack). In optical imaging techniques, image evaluation is the time-limiting factor.
In addition to optical imaging techniques, there are also methods of measurement using scanning probe microscopy (SPM). Scanning probe microscopy is a technique in which a measuring probe scans a sample to be analysed and for example records the topography of the sample. In this connection there is relative motion between the measuring probe and the sample, which is achieved by moving at least the measuring probe or at least the sample. Usually the relative motion is performed as a lateral movement. Additionally, there can also be relative motion in the vertical direction. One form of scanning probe microscopy is scanning force microscopy (SFM). In a scanning force microscope used for this, the measuring probe is designed as a cantilever, which carries a fine measuring tip. The SFM techniques include the AFM technique (AFM: atomic force microscopy).
An SPM measurement can be carried out as a slow, label-free imaging technique. On a cantilever measuring beam there is a suitable measuring tip, for example a ligand-coated particle. The measuring beam and the measuring tip form a measuring probe, which is also called a cantilever. For measurement, a relative motion is performed between the measuring probe and a sample to be analysed, which can for example be cells with a set of receptors corresponding to the ligand. Molecular interactions between the sample (receptors) and the measuring tip (ligand) can lead to a deflection of the measuring probe, which can be documented in particular as bending of the measuring beam.
The SPM technique finds application both in the analysis of molecular interactions between isolated proteins and in the analysis of for example receptor-ligand interactions on cell-cell, cell-particle or cell-substrate samples. The result of the analysis typically comprises a description of mechanical characteristics such as viscoelasticity, strength of adhesion and surface topology. Automation of the SPM technique has been considered, as disclosed for example in documents DE 10 2004 048971 and WO 2006/040025. These describe an automated set-up for measurement and evaluation by scanning microscopy.
If, during probe-microscopic analysis of a sample, there is lateral movement of the measuring probe relative to the sample, a surface image can be recorded with the corresponding mechanical-elastic properties of the sample. A disadvantage is that for imaging when using an AFM microscope, depending on the local resolution of measurement, about 10 min to 1 h is required for one cell. Therefore there are clear limitations on the use of the AFM technique for the industrial screening of active substances. To improve the time factor, the use of multi-cantilevers has been proposed (cf. DE 10 2007 023 435). Alternatively, the areal imaging property of the AFM technique is abandoned and the sample is only scanned at points, so that measurements of local interaction are carried out. This measurement principle is usually known as single cell force spectroscopy (SCFS). The mechanical properties that can be determined by AFM include for example elasticity, e.g. the elasticity of one cell, and measurement of the forces on cell-cell or cell-substrate contacts. With a suitable choice of measuring probe design, the sample can also be analysed electrically.
There are essential differences between the measurement of mechanical properties such as protein-protein interactions and measurements on cell-free preparations and cellular samples. These include the far larger height dimension of cells (z-height) and the discontinuous distribution of the cells. Typically, the cells do not form a uniform continuum on a cultivation substrate in vitro. Their height profile itself and the relative position of important cell organelles can vary, so too can the cell-cell distance and the cell shape. Therefore it is also important in single-cell force microscopy to employ special placement of the measuring tip. A property of confocal microscopy is that only an image segment limited in height to about 1 to 2 μm is obtained. With a larger height dimension of the object to be analysed, in certain circumstances it is important to record several camera images from different heights and evaluate them individually or combine them as a so-called z-stack for 3D illustration.
Furthermore, not every measurement of interaction in single-cell force microscopy is successful. This is strongly dependent on the initial biological situation. Thus, an adhesion frequency of <30% in force measurements (LFA-1/ICAM-1 on A39 cells) has been reported (Wojcikiewicz et al., Biol. Proced. Online 2004:6 1-9, Force and compliance measurements on living cells using atomic force microscopy). This effect can greatly increase the measurement time and the number of cells required. At the same time, however, single-cell force microscopy (SCFS) makes it possible, in contrast to bulk adhesion assays, to resolve various subpopulations and to observe the effect of active substances, for example inhibitors, at the level of the individual cell and even the individual molecule.
It should be noted that optical detection of cells at higher resolution, for example by means of fluorescence, typically requires thin glass, with a thickness of about 170 μm, as substrate. It is important to ensure that the reduced mechanical stability of the thin glass does not lead to vibrations, which can disturb the AFM measurement.